Contribuição ao estudo da diversidade do HIV-1 no Brasil: caracterização genotípica e fenotípica de amostras de HIV-1 obtidas em diferentes regiões geográficas

Made available in DSpace on 2017-10-30T15:42:11Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) jose_fernandez_ioc_dout_2000.pdf: 5430529 bytes, checksum: 26b12ad2dc0974b2410f6f75f4116604 (MD5)Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil.Desde o estabelecimento em cultura do primeiro isolado de HIV-1 no Brasil (Galvão- Castro e cols., 1987), já podíamos perceber um polimorfismo genético e biológico em amostras obtidas no início da epidemia de AIDS no país, indicando que esta heterogeneidade seria similar a descrita para o HIV-1 em outras partes do mundo (Couto-Fernandez e cols., 1992). Nos anos subsequentes, buscando um melhor detalhamento da estrutura genética destes isolados, entre outros, sequenciamos um fragmento genômico de aproximadamente 860pb correspondentes as regiões V3, V4, V5 e parte da gp41 do envelope viral de 6 amostras coletadas entre 1987 e 1989 (documento 1). A análise filogenética mostrou que estas amostras brasileiras eram do subtipo B, com níveis de diversidade variando entre 5,9 e 13, 1%. A análise das seqüências de aminoácidos da alça V3 revelou que 3 amostras possuíam o motivo GPGR no topo da alça, característico de isolados Norte Americanos/Europeus, enquanto que nas outras três existia uma substituição da prolina pelo triptofano (W), metionina (M) e fenilalalina (F), respectivamente. O grau de diversidade antigênica, avaliado pela reatividade sorológica de 114 soros obtidos de pacientes do Rio de Janeiro, coletados no período de 1990 a 1992, frente à diferentes peptídeos sintéticos subtipo-específicos, mostrou que 60,5% deles reagiram com peptídeos do subtipo B e 15,8% mostraram reatividade específica para o peptídeo Bra-cons (GWG). Um elevado percentual de soros (37,7%) não mostrou reatividade com os diferentes peptídeos usados. Buscando conhecer o espectro da diversidade do HIV-1 nos sítios selecionados para futuros testes de vacinas anti-HIV/AIDS, foi estabelecida a Rede Nacional de Laboratórios para Isolamento e Caracterização do HIV-1 no Brasil, que permitiu uma análise abrangente e sistematizada de amostras coletadas em diferentes regiões do país Estes estudos, realizados primeiramente em amostras do Rio de Janeiro (documento 2) e, subseqüentemente, de forma mais detalhada em amostras provenientes de três grandes cidades brasileiras (São Paulo, Rio de Janeiro e Belo Horizonte), permitiu a identificação de três subtipos genéticos do HIV-1, na proporção de 82,9% das amostras do subtipo B, 14,3% subtipo F e 2,9% subtipo C (documento 3). A variante B\2019\2019 foi identificada em 45% das amostras do subtipo B. A análise filogenética de 25 isolados virais utilizando seqüências genômicas correspondentes a região C2V3 do envelope viral, mostrou 100% de concordância com os resultados da análise da mobilidade de heteroduplexes - HMA. Não foi observada associação significativa entre os diferentes subtipos genéticos e propriedades biológicas in vitro. Confirmando dados anteriores, não foi demonstrada associação entre subtipos genéticos e perfis de soro-reatividade frente a peptídeos subtipoespecíficos ou neutralização heteróloga. Reconhecendo a existência de lacunas no conhecimento sobre os níveis de diversidade do HIV-1 em outras regiões do Brasil, fora do eixo Rio - São Paulo, realizamos a subtipagem genética do HIV-1 em amostras obtidas de diferentes populações de risco na cidade de Salvador-Bahia (documento 4) que, atualmente, concentra o segundo maior número de casos de AIDS na região nordeste. Neste estudo foram analisadas amostras de usuários de drogas injetáveis (UDI) e de indivíduos infectados por via sexual. No grupo dos UDI, 89,5% das amostras foram classificadas como subtipo B, 3% subtipo F e 7,5% mostrou um perfil B/F na análise pelo HMA. No grupo de transmissão sexual, 95% das amostras eram do subtipo B, 3,4% mostrou um perfil B/F e uma amostra o perfíl B/C/E no HMA O sequenciamento genômico das amostras com múltiplos perfis no HMA permitiu classificá-las filogenéticamente como sendo do subtipo B, enquanto que as amostras do subtipo F, se agruparam com amostras brasileiras referência do mesmo subtipo. A análise da reatividade sorológica frente à diferentes peptídeos sintéticos subtipo-específico mostrou níveis elevados de reatividade cruzada entre os diferentes peptídeos, não permitindo a discriminação sorológica dos diferentes subtipos do HIV-1 no Brasil, bem como da variante B´´. Ainda no referido estudo, não pudemos verificar associação entre os subtipos de HIV-1 e via de transmissão, sexo ou fator racial. Estimativas do Programa de AIDS das Nações Unidas (1999) calculam que nos próximos anos, vírus do subtipo não-B serão os principais responsáveis pelo surgimento de novos casos de AIDS, principalmente nos países em desenvolvimento. Neste contexto, buscando obter informações detalhadas sobre a estrutura molecular de vírus não-B circulante no Brasil, realizamos o sequenciamento molecular de uma amostra do subtipo D, recentemente identificada no Rio de Janeiro. A análise das seqüências de nucleotídeos da amostra D brasileira mostrou estreita relação filogenética com isolados africanos do mesmo subtipo (documento 5). Os resíduos de cisteínas se mostraram relativamente conservados, novos sítios de glicosilação e uma grande inserção foi detectada na região V1/V2 do envelope viral, enquanto que o domínio GPGQ estava presente no topo da alça V3. Nenhum evento de recombinação foi verificado no envelope e genes acessórios, contudo, a porção 5´do gene nef mostrou relação filogenética com vírus do subtipo B Concluindo, realizamos um estudo comparativo dos níveis de diversidade da região V3 das amostras de HIV-1 incluídas neste estudo, procurando determinar o envolvimento de substituições de aminoácidos básicos, bem como da distribuição de cargas nesta região, como determinante do fenótipo/tropismo viral (documento 6). Aparentemente, a variabilidade da região V3 não implicou em alteração significativa da sua estrutura espacial. Apesar de não existir diferença significativa da carga total, ponto isoelétrico (pI) e percentual de aminoácidos básicos da região V3, pudemos detectar uma maior concentração de cargas positivas nos isolados T-linfotrópicos, indutores de sincício, assim chamados SI. Não houve correlação dos parâmetros acima analisados com subtipo genético do HIV-1, fenótipo biológico ou distribuição geográficaSince the first isolation of a Brazilian HIV-1 strain (Galvão-Castro e cols., 1987) a genetic and biologic polymorphism of HIV-1 samples obtained at the beginning of the AIDS epidemic in Brazil was evident, similar to that described for HIV-1 in other regions of the world (Couto-Fernandez e cols., 1992). Subsequently, to better know the genomic structure of this and other isolates, we sequenced a 860-base pair envelope fragment including V3, V4, V5, and the beginning of gp41 of six Brazilian HIV-1 strains, isolated between 1987 and 1989. The phylogenetic analysis classified all Brazilian samples as genotype B, with interhost distances between 5.9 and 13,1%. The amino acid sequence analysis of the V3 loops revealed that three strains contained the North American/European GPGR motif at the top of the loop whereas in the other three strains proline (P) was substituted by tryptophan (W), methionine (M), or phenylalanine (F), respectively. The antigenic diversity was analyzed through the serological reactivity of 114 sera of HIV-1 infected patients from Rio de Janeiro (1990/1992), against subtype-specific HIV-1 synthetic peptides. Sixty-nine sera (60.5%) reacted with peptides belonging to genotype B and 15,8% had biding antibodies to the Bra-cons (GWG) peptide. A high number of sera (37,7%) had no antibodies to any of the V3 peptides tested. To better understand the spectrum of HIV-1 diversity in three potential HIV vaccine sites in Brazil, the Brazilian Network for HIV isolation and Characterization was established for a systematic surveillance of HIV variability in different geographic regions These studies were first performed in HIV-1 samples obtained from patients from Rio de Janeiro (document 2) and, subsequently more detailed analysis of isolates from three big Brazilian cities (São Paulo, Rio de Janeiro and Belo Horizonte), revealed the presence of three genetic subtypes of HIV-1 identified by HMA: B (in 82,9% of the samples), F (14,3%) and C (2,9%) [document 3]. The HIV-1 B\2019\2019 variant was identified in 45% of subtype B samples. Phylogenetic analysis based on the C2V3/env DNA sequence from all 25 specimens examined was 100% concordant with the heteroduplex mobility assay (HMA) results. No significant association was found between HIV-1subtypes and the mode of transmission or biological properties of the HIV-1 isolates. Consistent with previous results, no relationship between viral subtype and peptide ELISA seroreactivity or neutralization was evidenced. To expand the analysis to regions not belonging to the southeast Brazilian region, we investigated the distribution of HIV-1 genetic subtypes in different populations from Salvador \2013 Bahia (document 4), where the second major concentration of AIDS cases from the northeast Brazil is found nowdays. Blood samples from HIV-1 seropositive injecting drug users (IDUs) and individuals infected sexually, were analyzed using HMA. In the IDU group, 89,5% were classified as subtype B, 3% as subtype F, and 7,5% showed a B/F HMA profile In the sexual transmission (ST) group, 95% were identified as B subtype, 3,4% showed a B/F profile, and 1,6% a B/C/E HMA profile. All Brazilian samples that showed multiple reactivities in HMA analysis clustered on sequencing with North American/European HIV-1 B subtype isolates in the phylogenetic analysis, whereas the F subtypes clustered with F Brazilian HIV-1 isolates. Serologic reactivities of IDU\2019s sera were examined using a panel of synthetic V3 loop peptides representative of the different HIV-1 subtypes. A high level of cross-reactivity was observed against the different peptides, not permitting the serologic HIV-1 typing of the genetic subtypes circulating in Brazil, even of the B\2019\2019 variant. No difference in the serologic reactivity between F and B subtype plasma could be observed. In this study, no significant association was found between HIV-1 subtypes and the mode of transmission. For the coming decades the AIDS Program of the United Nations (UNAIDS, 1999) estimates that non-B subtypes of HIV-1 will be responsible for the majority of new AIDS cases in developing countries. In this context, we genetically characterized a HIV-1 subtype D strain recently identified in Rio de Janeiro, Brazil. The nucleotide sequence of the Brazilian subtype D clearly grouped with African HIV-1 viruses from the same subtype (document 5). The cysteine residues were relatively well conserved, new glycosilation sites and a big insertion was detected in the V1/V2 envelope region. However, the V3 diplayed the characteristic GPGQ motif in the top. No recombinant events were observed in the viral envelope and accessory genes. However, the 5\2019 region of the nef gene appears to be closer to subtype B sequences In conclusion, in order to evaluate the molecular diversity in the envelope V3 region of the Brazilian HIV-1 strains included in this study, we analyzed the patterns of amino acid substitutions of NSI-like and SI-like isolate sequences to verify the involvement of basic amino acid substitutions, as well as the distribution of charges in this region, as predictor of viral phenotype/tropism (document 6). Apparently, the V3 region diversity was not implied in significant changes in its spatial structure. Although no significant differences in the total charges, isoelectric points (pI) and percentages of basic amino acids in the V3 region of Brazilian strains, a higher concentration of positive charges in Tlymphotropic \201Csyncytium inducing\201D isolates could be detected. No correlation was observed between the parameters analyzed and HIV-1 genetic subtype, biologic phenotype or geographic distributio

Prevalence of HIV type 1 drug resistance mutations in treatment-naïve and experienced patients from resource-limited settings with universal access to antiretroviral therapy: a survey in two small Brazilian cities

Concerns have been raised that universal availability of antiretroviral agents in resource-limited settings might lead to the emergence and spread of resistant strains. We present the largest survey on human immunodeficiency virus type 1 (HIV-1) resistance among treatment-naïve and experienced patients followed in small, relatively underprivileged cities in Brazil with universal availability to standard of care antiretroviral combinations. Samples were collected between 2004 and 2006 from 95 patients followed in the cities of Saquarema and Santo Antonio de Pádua, state of Rio de Janeiro. A proviral fragment encompassing protease and reverse transcriptase (RT) regions was generated and drug susceptibility level was inferred. Among 50 strains from drug-naïve subjects, one (2%) had intermediate-level resistance to RT inhibitors. Among 38 patients on therapy as of sampling, 28 (73.7%) had plasma viral load (PVL) below detection limit (26 of whom without evidence of resistance mutations) and 11 (28.9%) harbored strains with reduced susceptibility. Only two strains harbored both protease and RT inhibitor mutations. Among seven patients who were off-treatment as of sampling, two (28.5%) harbored strains with reduced susceptibility to RT inhibitors. The relatively high frequency of undetectable PVL among patients on treatment and the overall low prevalence of resistance-associated mutations are reassuring. Continued surveillance, however, is necessary

Molecular and biological diversity of HIV-1 in Brazil.

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2012-10-22T19:08:59Z No. of bitstreams: 1 Couto-Fernandes JC Molecular and biological ....pdf: 353616 bytes, checksum: d613ed7fa2e770cdea12fb1f199f89db (MD5)Made available in DSpace on 2012-10-22T19:08:59Z (GMT). No. of bitstreams: 1 Couto-Fernandes JC Molecular and biological ....pdf: 353616 bytes, checksum: d613ed7fa2e770cdea12fb1f199f89db (MD5) Previous issue date: 1992Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilFundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Imunologia. Rio de Janeiro, RJ, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilTo determine the genomic polymorphism and biological properties present in HIV-1 Brazilian isolates, we analyzed five viral isolates obtained from patients residing in Rio de Janeiro (P1 and P5), São Paulo (P3) and Bahia (P2 and P4) states. For each viral isolate in vitro characteristics such as replication rate, syncytium-inducing capacity and cell death were observed in lymphoblastoid (H9, CEM and peripheral blood mononuclear cells) as well as monocytoid (U937) cells. In addition, the evaluation of the restriction fragment length polymorphism of these isolates was also performed using a panel of endonucleases such as Hind III, Bgl II, Sac I, Pst I, Kpn I and Eco RI. One of the isolates (P1), showed the highest phenotypic and genotypic divergence, when compared to others. The results found suggest a HIV heterogeneity in Brazil similar to that already described in other regions of the world

HIV-1 subtyping in Salvador, Bahia, Brazil: a city with African sociodemographic characteristics

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2012-10-04T20:34:19Z No. of bitstreams: 1 Couto-Fernandes HIV_1_Subtyping_in_Salvador,_Bahia,_Brazil__A_City.11[1].pdf: 4269771 bytes, checksum: c4d52085f0cd1c6b1513fbae32f04c21 (MD5)Made available in DSpace on 2012-10-04T20:34:19Z (GMT). No. of bitstreams: 1 Couto-Fernandes HIV_1_Subtyping_in_Salvador,_Bahia,_Brazil__A_City.11[1].pdf: 4269771 bytes, checksum: c4d52085f0cd1c6b1513fbae32f04c21 (MD5) Previous issue date: 1999Fundação Oswaldo Cruz. Departamento de Imunologia. Rio de Janeiro, RJ, BrasilFundação Oswaldo Cruz. Departamento de Imunologia. Rio de Janeiro, RJ, BrasilFundação Oswaldo Cruz. Departamento de Imunologia. Rio de Janeiro, RJ, BrasilUniversidade Federal do Rio de Janeiro. Departamento de Genética. Rio de Janeiro, RJ, BrasilUniversidade Federal da Bahia. Centro de Tratamento do Abuso de Drogas. Salvador, BA, BrasilUniversidade Federal da Bahia. Laboratorio de Retrovirologia Prof. Edgard Santos Hospital. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilTo investigate the prevalence of the HIV-1 subtypes in different populations from Salvador, Bahia, Brazil, blood samples from 72 HIV-1-seropositive injecting drug users (IDUs) and 62 individuals infected sexually were analyzed using the heteroduplex mobility assay (HMA). In the IDU group, 89.5% were classified as subtype B, 3% as subtype F, and 7.5% showed a B/F HMA profile. In the sexual transmission (ST) group, 95% were identified as B subtype, 3.4% showed a B/F profile, and 1.6% a B/C/E HMA profile. All Brazilian samples that showed multiple reactivities in the HMA analysis clustered on sequencing with B North American/ European HIV-1 isolates in the phylogenetic analysis, whereas the F subtypes clustered with F Brazilian HIV-I isolates. Serologic reactivities of IDU's sera were examined using a panel of synthetic V3 loop peptides representative of the different HIV-1 subtypes. No difference in serologic reactivity between F and B subtype plasma could be observed. Predominance of HIV-I subtype B was identified in both study groups, whereas subtype F was detected only among IDUs in a frequency lower than described for other Brazilian regions

HIV-1 Diversity and Drug Resistance in Treatment-Naïve Children and Adolescents from Rio de Janeiro, Brazil

The human immunodeficiency virus type 1 (HIV-1) can be transmitted via parenteral, sexual, or vertical exposure routes. The number of HIV-1 cases detected yearly in children and adolescents in Brazil did not decrease over the last decade, representing ~5% of total cases described in the country. In recent years, the HIV-1 diversity and the prevalence of transmitted drug resistance mutations (TDRM) are moving toward a marked increase. In this study, we retrospectively evaluated the diversity of HIV-1 subtypes and the TDRM prevalence in 135 treatment-naïve HIV-1 vertically infected children and adolescents born in between 1993 and 2012. These children were assessed in either 2001–2007 or 2008–2012 when they were 0 to 17 years old. The individuals assessed in 2001–2007 (n = 38) had median CD4+ T cell counts of 1218 cells/mm3 (IQR: 738–2.084) and median HIV-1 plasma viral load of 4.18 log10 copies/mL (IQR: 3.88–4.08). The individuals (n = 97) evaluated in 2008–2012 showed median CD4+ T cell counts of 898.5 cells/mm3 (IQR: 591.3–1.821) and median HIV-1 plasma viral load of 4.69 log10 copies/mL (IQR: 4.26–5.33). A steady decrease in the median CD4 T+ cell counts was observed with age progression, as expected. The majority HIV-1 pol sequences (87%) were classified as pure HIV-1 subtypes (77% subtype B, 9% subtype F1 and 1.5% subtype C), while 13% of sequences were classified as recombinants (CRF45_cpx, n = 4; CRF28/29_BF1, n = 2; CRF02_AG, n = 1; CRF40_BF1, n = 1, CRF99_BF1, n = 1, URF_BF1, n = 8). The overall prevalence of TDRM was 14% (19/135), conferring resistance to the nucleoside reverse transcriptase inhibitors (NRTI, 13/135–9.6%), non-nucleoside reverse transcriptase inhibitors (NNRTI, 8/135–5.9%), and protease inhibitors (PI, 2/135–1.5%). The main TDRM observed for NNRTI was the K103N (n = 8), while the mutations T215I/Y/D/E (n = 7) and M184V (n = 4) were the main TDRM for NRTI. Only two TDRM were observed for PI in one individual each (M46I and V82A). Most TDRM were found in the HIV-1 subtype B (84%) sequences. This study reveals an HIV-1 epidemic with high diversity and moderate prevalence of TDRM in the pediatric population of Rio de Janeiro, indicating the existence of possible problems in the clinical management of prophylactic therapy to prevent mother-to-child transmission and future treatment options for the affected children

Genetic diversity and drug resistance of HIV-1 among infected pregnant women newly diagnosed in Luanda, Angola

Project Pró-África CNPq n˚ 440145/2015-5Monitoring genetic diversity and drug resistance mutations (DRMs) is critical for understanding HIV epidemiology. Here, we report HIV-1 genetic diversity and DRMs in blood samples from 42 HIV-positive pregnant women naive to antiretroviral therapy (ART), in Luanda. The samples were subjected to nested-PCR, followed by sequencing of the HIV-1 pol gene, targeting the protease and reverse transcriptase fragments. HIV-1 diversity was analyzed using the REGA HIV-1 subtyping tool and DRMs were identified using the Calibrated Population Resistance tool. A total of 34 sequences were obtained. The data revealed wide HIV-1 subtypes heterogeneity, with subtype C (38%, 13/34) the most frequent, followed by the subtypes F1 (18%, 6/34), A1 (9%, 3/34), G (9%, 3/34), D (6%, 2/34) and H (3%, 1/34). In addition, recombinants strains were detected, with CRF02_AG (6%, 2/34) the most frequent, followed by CRF37_cpx, F1/C, A1/G, and H/G, all with 3% (1/34). A total of 6/34 (18%) of the sequences presented DRMs. The non-nucleoside reverse transcriptase inhibitors presented 15% (5/34) of resistance. Moreover, 1/34 (3%) sequence presented resistance against both non-nucleoside reverse transcriptase inhibitors and nucleoside reverse transcriptase inhibitors, simultaneously. Despite the small sample size, our results suggest the need to update currently used ART regimens. Surveillance of HIV-1 subtypes and DRMs are necessary to understand HIV epidemiology and to guide modification of ART guidelines in Angola.info:eu-repo/semantics/publishedVersio

Single Nucleotide Polymorphisms in Cellular Drug Transporters Are Associated with Intolerance to Antiretroviral Therapy in Brazilian HIV-1 Positive Individuals

<div><p>Adverse reactions are the main cause of treatment discontinuation among HIV+ individuals. Genes related to drug absorption, distribution, metabolism and excretion (ADME) influence drug bioavailability and treatment response. We have investigated the association between single nucleotide polymorphisms (SNPs) in 29 ADME genes and intolerance to therapy in a case-control study including 764 individuals. Results showed that 15 SNPs were associated with intolerance to nucleoside and 11 to non-nucleoside reverse transcriptase inhibitors (NRTIs and NNRTIs), and 8 to protease inhibitors (PIs) containing regimens under alpha = 0.05. After Bonferroni adjustment, two associations remained statistically significant. SNP rs2712816, at <i>SLCO2B1</i> was associated to intolerance to NRTIs (OR_GA/AA = 2.37; p = 0.0001), while rs4148396, at <i>ABCC2</i>, conferred risk of intolerance to PIs containing regimens (OR_CT/TT = 2.64; p = 0.00009). Accordingly, haplotypes carrying rs2712816A and rs4148396T alleles were also associated to risk of intolerance to NRTIs and PIs, respectively. Our data reinforce the role of drug transporters in response to HIV therapy and may contribute to a future development of personalized therapies.</p></div